Antibody Induction Directed against the Tumor‐Associated MUC4 Glycoprotein

Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti‐tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor‐associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma. The aberrant glycosylation of MUC4 in tumor cells results in an exposure of its peptide backbone and the formation of tumor‐associated glycopeptide antigens. Due to the low immunogenicity of these endogenous structures, their conjugation with immune stimulating peptide or protein carriers are required. In this study, MUC4 tandem‐repeat glycopeptides were conjugated to the tetanus toxoid and used for vaccination of mice. Immunological evaluations showed that our MUC4‐based vaccines induced very strong antigen‐specific immune responses. In addition, antibody binding epitope analysis on glycopeptide microarrays, were demonstrating a clear glycosylation site dependence of the induced antibodies.     

Lactose‐Functionalized Dendrimers Arbitrate the Interaction of Galectin‐3/MUC1 Mediated Cancer Cellular Aggregation

By using lactose‐functionalized poly(amidoamine) dendrimers as a tunable multivalent platform, we studied cancer cell aggregation in three different cell lines (A549, DU‐145, and HT‐1080) with galectin‐3. We found that small lactose‐functionalized G(2)‐dendrimer 1 inhibited galectin‐3‐induced aggregation of the cancer cells. In contrast, dendrimer 4 (a larger, generation 6 dendrimer with 100 carbohydrate end groups) caused cancer cells to aggregate through a galectin‐3 pathway. This study indicates that inhibition of cellular aggregation occurred because 1 provided competitive binding sites for galectin‐3 (compared to its putative cancer cell ligand, TF‐antigen on MUC1). Dendrimer 4 , in contrast, provided an excess of ligands for galectin‐3 binding; this caused crosslinking and aggregation of cells to be increased.     

Merging the Versatile Functionalities of Boronic Acid with Peptides

Peptides inherently feature the favorable properties of being easily synthesized, water-soluble, biocompatible, and typically non-toxic. Thus, boronic acid has been widely integrated with peptides with the goal of discovering peptide ligands with novel biological activities, and this effort has led to broad applications. Taking the integration between boronic acid and peptide as a starting point, we provide an overview of the latest research advances and highlight the versatile and robust functionalities of boronic acid. In this review, we summarize the diverse applications of peptide boronic acids in medicinal chemistry and chemical biology, including the identification of covalent reversible enzyme inhibitors, recognition, and detection of glycans on proteins or cancer cell surface, delivery of siRNAs, development of pH responsive devices, and recognition of RNA or bacterial surfaces. Additionally, we discuss boronic acid-mediated peptide cyclization and peptide modifications, as well as the facile chemical synthesis of peptide boronic acids, which paved the way for developing a growing number of peptide boronic acids.     

Effect of Ganglioside GM3 Synthase Gene Knockout on the Glycoprotein N‐Glycan Profile of Mouse Embryonic Fibroblast

The structural and clinical significance of cellular glycoproteins and glycosphingolipids (GSLs) are often separately discussed. Considering the biosynthetic pathway of glycoconjugates, glycans of cell‐surface glycoproteins and GSLs might partially share functions in maintaining cellular homeostatis. The purpose of this study is to establish a general and comprehensive glycomics protocol for cellular GSLs and N‐glycans of glycoproteins. To test the feasibility of a glycoblotting‐based protocol, whole glycans released both from GSLs and glycoproteins were profiled concurrently by using GM3 synthase‐deficient mouse embryonic fibroblast GM3(?/?). GM3(?/?) cells did not synthesize GM3 or any downstream product of GM3 synthase. Instead, expression levels of o‐series gangliosides involving GM1‐b and GD1‐α increased dramatically, whereas a‐/b‐series gangliosides were predominantly detected in wild‐type (WT) cells. We also discovered that glycoprotein N‐glycan profiles of GM3(?/?) cells are significantly altered as compared to WT cells, although GM3 synthase is responsible only for GSLs synthesis and is not associated with glycoprotein N‐glycan biosynthesis. The present approach allows for high‐throughput profiling of cellular glycomes enriched by different classes of glycoconjugates, and our results demonstrated that gene knockout of the enzymes responsible for GSL biosynthesis significantly influences the N‐glycans of glycoproteins.     

Selective detection of dopamine using a functionalised polyaniline composite electrode

The behaviour of a poly (aniline boronic acid) (PABA) modified glassy carbon electrode (GCE) for the detection of dopamine (DA) in the presence of excess of ascorbic acid (AA) using cyclic voltammetry (CV) and differential pulse voltammetry (DPV) techniques is investigated. On bare GCE, both DA and AA are oxidized at ~0.16 V, whereas on PABA modified GCE they are oxidized at 0.2 and 0.054 V, respectively. Though PABA favours DA oxidation through ester formation with boronic acid motif, the AA oxidation is also promoted by polyaniline backbone through the involvement of AA in the redox of polyaniline. Since both DA and AA undergo oxidation at closely spaced potentials at a PABA electrode, Nafion®-incorporation into the PABA film was examined for selective determination of DA in the presence of AA. The selectivity was due to accumulation of DA on the electrode surface through ester formation with the boronic acid group and suppression of AA oxidative current through charge discrimination by Nafion.     

Boronic Acid-Based Approach for Separation and Immobilization of Glycoproteins and Its Application in Sensing

Glycoproteins influence a broad spectrum of biological processes including cell-cell interaction, host-pathogen interaction, or protection of proteins against proteolytic degradation. The analysis of their glyco-structures and concentration levels are increasingly important in diagnosis and proteomics. Boronic acids can covalently react with cis-diols in the oligosaccharide chains of glycoproteins to form five- or six-membered cyclic esters. Based on this interaction, boronic acid-based ligands and materials have attracted much attention in both chemistry and biology as the recognition motif for enrichment and chemo/biosensing of glycoproteins in recent years. In this work, we reviewed the progress in the separation, immobilization and detection of glycoproteins with boronic acid-functionalized materials and addressed its application in sensing.     

Synthetic MUC1 Antitumor Vaccine with Incorporated 2,3‐Sialyl‐T Carbohydrate Antigen Inducing Strong Immune Responses with Isotype Specificity

The endothelial glycoprotein MUC1 is known to underlie alterations in cancer by means of aberrant glycosylation accompanied by changes in morphology. The heavily shortened glycans induce a collapse of the peptide backbone and enable accessibility of the latter to immune cells, rendering it a tumor‐associated antigen. Synthetic vaccines based on MUC1 tandem repeat motifs, comprising tumor‐associated 2,3‐sialyl‐T antigen, conjugated to the immunostimulating tetanus toxoid, are reported herein. Immunization with these vaccines in a simple water/oil emulsion produced a strong immune response in mice to which stimulation with complete Freund's adjuvant (CFA) was not superior. In both cases, high levels of IgG1 and IgG2a/b were induced in C57BL/6 mice. Additional glycosylation in the immunodominant PDTRP domain led to improved binding of the induced antisera to MCF‐7 breast tumor cells, compared with that of the monoglycosylated peptide vaccine.     

Imparting affinity sites for adenosine triphosphate on the surface of polyurethane through molecular imprinting

A simple methodology for imparting affinity sites for adenosine triphosphate (ATP) on the surface of polyurethane (PU) film is discussed. It is widely known that, through oxidation, a thin layer conjugated polymer can be coated onto many polymeric substrates. This possibility is explored in this report. 3‐Amino phenyl boronic acid was polymerized in the presence of ATP as template using ammonium per sulfate. A thin layer of polyamino phenyl boronic acid was coated onto the surface of PU film immersed in the solution during the reaction. The coated layer showed remarkably high affinity toward ATP and equilibrium adsorption was attained rapidly in contrast to free standing molecularly imprinted polymers in which equilibrium adsorption is normally attained after several hours. The imprinted layer also showed a high degree of selectivity toward ATP compared to adenosine diphosphate (ADP). The approach is simple and affinity sites for any water soluble molecules can virtually be created on the surface of polymers of interest. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 94: 2088–2090, 2004     

Synthesis and Evaluation of the Anticancer and Trypanocidal Activities of Boronic Tyrphostins

Molecules containing an (cyanovinyl)arene moiety are known as tyrphostins because of their ability to inhibit proteins from the tyrosine kinase family, an interesting target for the development of anticancer and trypanocidal drugs. In the present work, (E)‐(cyanovinyl)benzeneboronic acids were synthesized by Knoevenagel condensations without the use of any catalysts in water through a simple protocol that completely avoided the use of organic solvents in the synthesis and workup process. The in vitro anticancer and trypanocidal activities of the synthesized boronic acids were also evaluated, and it was discovered that the introduction of the boronic acid functionality improved the activity of the boronic tyrphostins. In silico target fishing with the use of a chemogenomic approach suggested that tyrosine‐phosphorylation‐regulated kinase 1a (DYRK1A) was a potential target for some of the designed compounds.     

Truncated O-Glycan-Bearing MUC16 Enhances Pancreatic Cancer Cells Aggressiveness via α4β1 Integrin Complexes and FAK Signaling

Elevated levels of Mucin-16 (MUC16) in conjunction with a high expression of truncated O-glycans is implicated in playing crucial roles in the malignancy of pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms by which such aberrant glycoforms present on MUC16 itself promote an increased disease burden in PDAC are yet to be elucidated. This study demonstrates that the CRISPR/Cas9-mediated genetic deletion of MUC16 in PDAC cells decreases tumor cell migration. We found that MUC16 enhances tumor malignancy by activating the integrin-linked kinase and focal adhesion kinase (ILK/FAK)-signaling axis. These findings are especially noteworthy in truncated O-glycan (Tn and STn antigen)-expressing PDAC cells. Activation of these oncogenic-signaling pathways resulted in part from interactions between MUC16 and integrin complexes (α4β1), which showed a stronger association with aberrant glycoforms of MUC16. Using a monoclonal antibody to functionally hinder MUC16 significantly reduced the migratory cascades in our model. Together, these findings suggest that truncated O-glycan containing MUC16 exacerbates malignancy in PDAC by activating FAK signaling through specific interactions with α4 and β1 integrin complexes on cancer cell membranes. Targeting these aberrant glycoforms of MUC16 can aid in the development of a novel platform to study and treat metastatic pancreatic cancer.     

Synthetic vaccines from tumor-associated glycopeptide antigens

The aberrantly glycosylated and extensively over-expressed membrane-bound mucin MUC1 glycoprotein forms a tumor specific epitope on the surface of epithelial cells. Using defined synthetic glycopeptide structures consisting of the MUC1 tandem repeat region for immunization, antibodies selectively binding to tumor cell surface should be induced. Recent examples of synthetic vaccines directed against the O-glycosylated MUC1 tandem repeats and their immunological evaluation will be given here. These include synthetic MUC1 glycopeptides conjugated to immunostimulants, such as T-cell peptide epitopes, immune carrier proteins or lipid immunostimulants.     

Exploring Molecular Contacts of MUC1 at CIN85 Binding Interface to Address Future Drug Design Efforts

The modulation of protein-protein interactions (PPIs) by small molecules represents a valuable strategy for pharmacological intervention in several human diseases. In this context, computer-aided drug discovery techniques offer useful resources to predict the network of interactions governing the recognition process between protein partners, thus furnishing relevant information for the design of novel PPI modulators. In this work, we focused our attention on the MUC1-CIN85 complex as a crucial PPI controlling cancer progression and metastasis. MUC1 is a transmembrane glycoprotein whose extracellular domain contains a variable number of tandem repeats (VNTRs) regions that are highly glycosylated in normal cells and under-glycosylated in cancer. The hypo-glycosylation fosters the exposure of the backbone to new interactions with other proteins, such as CIN85, that alter the intracellular signalling in tumour cells. Herein, different computational approaches were combined to investigate the molecular recognition pattern of MUC1-CIN85 PPI thus unveiling new structural information useful for the design of MUC1-CIN85 PPI inhibitors as potential anti-metastatic agents.     

Immune and Anticancer Responses Elicited by Fully Synthetic Aberrantly Glycosylated MUC1 Tripartite Vaccines Modified by a TLR2 or TLR9 Agonist

The mucin MUC1 is overexpressed and aberrantly glycosylated by many epithelial cancer cells manifested by truncated O‐linked saccharides. Although tumor‐associated MUC1 has generated considerable attention because of its potential for the development of a therapeutic cancer vaccine, it has been difficult to design constructs that consistently induce cytotoxic T‐lymphocytes (CTLs) and ADCC‐mediating antibodies specific for the tumor form of MUC1. We have designed, chemically synthesized, and immunologically examined vaccine candidates each composed of a glycopeptide derived from MUC1, a promiscuous T_helper peptide, and a TLR2 (Pam₃CysSK₄) or TLR9 (CpG‐ODN 1826) agonist. It was found that the Pam₃CysSK₄‐containing compound elicits more potent antigenic and cellular immune responses, resulting in a therapeutic effect in a mouse model of mammary cancer. It is thus shown, for the first time, that the nature of an inbuilt adjuvant of a tripartite vaccine can significantly impact the quality of immune responses elicited against a tumor‐associated glycopeptide. The unique adjuvant properties of Pam₃CysSK₄, which can reduce the suppressive function of regulatory T cells and enhance the cytotoxicity of tumor‐specific CTLs, are likely responsible for the superior properties of the vaccine candidate 1 .     

A TR‐FRET‐Based Functional Assay for Screening Activators of CARM1

Epigenetics is an emerging field that demands selective cell‐permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator‐associated arginine methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that twofold overexpression of CARM1 in MCF7 breast cancer cells increased the expression of ERα‐target genes involved in differentiation and reduced cell proliferation, thus leading to the hypothesis that activating CARM1 by chemical activators might be therapeutically effective in breast cancer. Selective, potent, cell‐permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell‐based, time‐resolved (TR) FRET assay that uses poly(A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen TR‐FRET assay uses MCF7 cells expressing GFP‐PABP1 fusion protein through BacMam gene delivery system, methyl‐PABP1 specific antibody, and terbium‐labeled secondary antibody. This assay has been validated as reflecting the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR‐FRET platform serves as a generic tool for functional screening of cell‐permeable, chemical modulators of CARM1 for elucidation of its in vivo functions.     

Impedimetric detection of dopamine on poly(3-aminophenylboronic acid) modified skeleton nickel electrodes

Detection of biologic compounds in particular dopamine is usually based on the complexation between boronic acid groups and diols. For this reason the development of new sensors based on direct monitoring of boronic acid–diol complexation is attractive. A measurable electric response due to a change in the dopamine concentration can be achieved on electrodes modified with boronic groups. In this work a modified electrode has been obtained by electropolymerization of 3-aminophenylboronic acid in aqueous solutions on a preformed polyaniline layer electrochemically deposited on smooth and skeleton nickel electrodes. The modified electrodes have been tested as impedimetric sensors for the detection of dopamine in aqueous phosphate buffer at pH = 7.4. Both sensors gave a linear response for dopamine concentrations between 10^?5 and 10^?10 mol L^?1. Poly(3-aminophenylboronic acid) modified skeleton nickel electrode has the advantage of an increased specific surface area, that lead to a high density of boronic acid groups and hence to a better sensitivity.     

Synthesis and Immunological Evaluation of a Multicomponent Cancer Vaccine Candidate Containing a Long MUC1 Glycopeptide

A fully synthetic MUC1‐based cancer vaccine was designed and chemically synthesized containing an endogenous helper T‐epitope (MHC class II epitope). The vaccine elicited robust IgG titers that could neutralize cancer cells by antibody‐dependent cell‐mediated cytotoxicity (ADCC). It also activated cytotoxic T‐lymphocytes. Collectively, the immunological data demonstrate engagement of helper T‐cells in immune activation. A synthetic methodology was developed for a penta‐glycosylated MUC1 glycopeptide, and antisera of mice immunized by the new vaccine recognized such a structure. Previously reported fully synthetic MUC1‐based cancer vaccines that elicited potent immune responses employed exogenous helper T‐epitopes derived from microbes. It is the expectation that the use of the newly identified endogenous helper T‐epitope will be more attractive, because it will activate cognate CD4⁺ T‐cells that will provide critical tumor‐specific help intratumorally during the effector stage of tumor rejection and will aid in the generation of sustained immunological memory.     

Development of an electrochemical‐surface plasmon dual biosensor based on carboxylated conducting polymer thin films

A biosensing platform based on the covalent attachment of biomolecules on electropolymerized carboxylated conducting polymers, poly(3‐aminobenzoic acid) and poly(3‐pyrrole carboxylic acid), were developed for the selective simultaneous detection of two biomolecules using electrochemical‐surface plasmon resonance (EC–SPR) spectroscopy. The surface morphology of the developed biosensors was studied by scanning electron microscopy and atomic force microscopy. The EC–SPR dual biosensor was developed for the label‐free, simultaneous, and selective detection of glucose and human immunoglobulin G (IgG). A change in current density was clearly observed after the injection of glucose, whereas a change in SPR reflectivity was clearly observed after the injection of human IgG. The present work demonstrates the potential of this biosensing platform for real sample analysis in the future. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 45641.     

A new thermosensitive fluorescent probe for diol sensing: Poly(N-isopropylacrylamide-co-vinylphenylboronic acid)-alizarin red S complex

The reversible complex formation between newly synthesized temperature sensitive copolymer, N-isopropylacrylamide-co-4-vinylphenylboronic acid and diol carrying molecules was first investigated by fluorescence spectroscopy. This investigation resulted in a development of a temperature sensitive-fluorescent probe used for quantitative determination of diol carrying molecules. In the first stage of competitive assay developed in this study, the receptor (i.e. boronic acid groups of temperature sensitive copolymer) and the reporter (a catechol dye, alizarin red S) associated at the selected pH and temperature to give a fluorescent cyclic boronate ester. The stimuli–responsive character of copolymer allowed to control the formation of fluorescent complex by adjusting the temperature. The maximum intensity was observed at 660 nm in the emission spectrum of the cyclic boronate ester. The fluorescent receptor–reporter complex (i.e. cyclic borate ester formed from boronic acid and ARS) was then partly dissociated in the presence of diol carrying guest due to the formation of another non-fluorescent complex between copolymer and guest. Various agents from different material groups, fructose, ascorbic acid, β-nicotinamide adenine dinucleotide (β-NAD) and horse radish peroxidase (HRP) were used as target diol carrying agents. The decrease in fluorescent intensity due to the dissolution of the receptor–reporter complex is proportional to the concentration of target diol carrying agent.     

Solvent extraction and purification of sugars from hemicellulose hydrolysates using boronic acid carriers

Research was performed to determine whether it was technically feasible to use boronic acid extractants to purify and concentrate the sugars present in hemicellulose hydrolysates. Initially, five types of boronic acids (phenylboronic acid, 3,5‐dimethylphenylboronic acid, 4‐tert‐butylphenylboronic acid, trans‐β‐styreneboronic acid or naphthalene‐2‐boronic acid) dissolved in an organic diluent (Shellsol^® 2046 or Exxal^® 10) containing the quaternary amine Aliquat^® 336 were tested for their ability to extract sugars (fructose, glucose, sucrose and xylose) from a buffered, immiscible aqueous solution. Naphthalene‐2‐boronic acid was found to give the greatest extraction of xylose regardless of which diluent was used. Trials were then conducted to extract xylose and glucose from solutions derived from the dilute acid hydrolysis of sugar cane bagasse and to then strip the loaded organic solutions using an aqueous solution containing hydrochloric acid. This produced a strip solution in which the xylose concentration had been increased over 7× that of the original hydrolysate while reducing the concentration of the undesirable acid‐soluble lignin by over 90%. Hence, this process can be exploited to produce high concentration xylose solutions suitable for direct fermentation. Copyright © 2004 Society of Chemical Industry     

氢供体组与辣根过氧化酶活性光谱研究

对邻苯二酚-苯胺和苯酚-氨基安替吡啉构成的两组测定HRP酶活性的体系进行了研究和比较。紫外可见光光谱和高效液相色谱分析表明,在HRP催化过程中,邻苯二酚-苯胺氢供体对会生成为一种粉红色产物,该产物最大吸收波长在510咖,可用于HRP的活性测量。且与常规使用的苯酚-氨基安替吡啉氢供体对的情况相比,具有更高的灵敏度、更低的检测限值和很好的重复性,在对HRP分别进行20次酶活测量,所得相对标准偏差仅为2．9％。使用邻苯二酚-苯胺氢供体对的方法可计算得到酶动力学参数Km（12．5mM）和Vmax（12．2mMmin^-1mg^-1）。